Since the discovery of the structure of nucleic acid molecules (DNA and RNA) in the 1950's, research into the structure, function, and use of these molecules has increased dramatically. In particular, a tremendous amount of research has been undertaken in order to utilize these molecules to diagnose and treat disease. In order however to obtain sufficient quantities of nucleic acids for use in experiments, and for diagnosis and treatment of disease, it is first necessary to purify and or isolate substantial quantities of nucleic acid molecules. For example, in the field of gene therapy, patients can be vaccinated or treated with nucleic acid molecules in order to protect against, or, remedy a disease (see, e.g., PCT publication WO 95/07994). However, preparation of large-quantities of purified, pharmaceutical-grade nucleic acid molecules is presently time-consuming and costly.
More specifically, while many methods exist for the purification of nucleic acid molecules, such methods are often limited when the nucleic acid molecules are to be produced for therapeutic purposes, since they must be prepared free of any contaminants such as toxic compounds and antigenic molecules. Prior art methods of DNA purification often utilized highly toxic chemicals such as ethidium bromide, phenol, chloroform, and cesium chloride (Sambrook, et al., 1992, Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor, N.Y.). Additionally, many methods involving both physical or chemical lysis often relied upon centrifugation to remove cellular debris, a step which is undesirable because of the difficulty in maintaining sterility. Furthermore, procedures using alkali lysis (Bimboim and Doly, Nucleic Acids Res. 7;1513, 1979) may lead to the loss of a significant amount of DNA through degradation caused by the increase in pH.
The present invention provides methods for producing purified nucleic acid molecules that are suitable for use in pharmaceutical applications, and on a large scale. Such methods do not require traditional techniques such as toxic extractants, mutagenic reagents, or steps such as centifugation. The present invention also provides other, related advantages.